Abstract

A survey for antibiotic-resistant (AR) Escherichia coli in wastewater was undertaken by collecting samples from primary clarifiers and secondary effluents from seven geographically dispersed US wastewater treatment plants (WWTPs). Samples were collected at each WWTP in cool and summer months and cultured using selective media. The resulting isolates were characterized for resistance to imipenem, ciprofloxacin, cefotaxime, and ceftazidime, presence of carbapenemase and extended-spectrum beta-lactamase (ESBL) genes, and phylogroups and sequence types (STs). In total, 322 AR E. coli isolates were identified, of which 65 were imipenem-resistant. Of the 65 carbapenem-resistant E. coli (CREC) isolates, 62% were positive for more than one and 31% were positive for two or more of carbapenemase and ESBL genes targeted. The most commonly detected carbapenemase gene was blaVIM (n = 36), followed by blaKPC (n = 2). A widespread dispersal of carbapenem-resistant STs and other clinically significant AR STs observed in the present study suggested the plausible release of these strains into the environment. The occurrence of CREC in wastewater is a potential concern because this matrix may serve as a reservoir for gene exchange and thereby increase the risk of AR bacteria (including CR) being disseminated into the environment and thence back to humans.

This article has been made Open Access thanks to the generous support of a global network of libraries as part of the Knowledge Unlatched Select initiative.

INTRODUCTION

Escherichia coli, a member of the family Enterobacteriaceae, is the predominant enterobacterial gut resident in humans and other mammals and is commonly used as an indicator of fecal pollution in source, drinking, and recreational waters (Elmund et al. 1999; Edberg et al. 2000; Odonkor & Ampofo 2013). Antibiotic resistance, including to extended-spectrum cephalosporins (ESCs) and carbapenems, is increasingly prevalent among Enterobacteriaceae. According to the Centers for Disease Control and Prevention (CDC), prior to 2002 less than 1% of E. coli clinical isolates were ESC-resistant or produced extended-spectrum β-lactamases (ESBLs), whereas in a recent report approximately 22% of clinical E. coli isolates were ESC-resistant (Weiner et al. 2016). Prior to 2010, reports of carbapenem-resistant Enterobacteriaceae (CRE) infections were uncommon in the USA; however, according to the CDC's National Healthcare Surveillance Network, the percentage of CRE isolates identified from hospital acquired infections ranged from 3.3 to 10.9%, depending on the site of infection (Weiner et al. 2016).

Historically, carbapenem antibiotics have been effective against multi-drug-resistant Gram-negative bacilli and a mainstay of therapy for infections due to such organisms. Carbapenem-resistant E. coli (CREC) is increasingly prevalent, mainly due to the emergence of novel carbapenemases. Currently, the three carbapenemases categorized as being most important are Klebsiella pneumoniae carbapenemase (KPC; Ambler class A), certain metalloenzymes (VIM, NDM; Ambler class B), and OXA-type enzymes (e.g., OXA-48; Ambler class D) (Nordmann et al. 2012). The associated carbapenemase genes are frequently found on mobile genetic elements and have the potential to spread to other Gram-negative bacteria (Nordmann et al. 2012).

Although CREC have been recognized primarily in health care settings, investigators at the US Environmental Protection Agency (EPA) proposed that antimicrobial resistance surveillance involving sewage isolates could be informative as to the occurrence and dissemination of specific clonal groups or sequence types within a given community or population (Boczek et al. 2007). wastewater treatment plants (WWTPs) typically use a multi-stage treatment approach, consisting of preliminary, primary, and secondary treatment with effluent disinfection. Primary treatment occurs when wastewater received by the treatment plant is collected into settling basins, where solids and grease are removed from the liquid portion using screens and gravity. Such processes are successful at removing approximately 60% of suspended solids from the wastewater. This primary effluent is then subjected to some type of secondary treatment, typically involving biological treatment to further breakdown the organic material in the wastewater. Following secondary treatment, the effluent is disinfected either with a chemical oxidizing agent, such as chlorine, or a physical treatment such as exposure to UV radiation (Manual of Practice 8; Design of Wastewater Treatment Plants 1998; Boczek et al. 2010).

This study is follow-on research to preliminarily survey wastewater, specifically primary and secondary effluents, from seven locations in the USA for antibiotic-resistant (AR) E. coli to ESCs and carbapenem, to characterize the resulting isolates for the presence of carbapenemase and ESBL genes and identify sequence types that have been associated with extraintestinal infections in humans.

MATERIALS AND METHODS

Study sites, sample collection and titer determination

Seven geographically dispersed WWTPs in the continental US were selected opportunistically for sampling. Permission was granted by WWTP operators on the condition of plant anonymity. The WWTPs were located in New Jersey, Maryland, Ohio, Texas, Colorado, northern California, and southern California, and served four urban and three rural/suburban areas. All participating plants used conventional activated sludge for secondary treatment of primary clarified effluents.

Samples were collected at each WWTP once during cooler months (November 2012 or April 2013) and once during the summer (July 2013 or August 2013), for a total of 28 samples, four per WWTP. Each sample consisted of either 1 liter from the primary clarifier effluent or an effluent from secondary treatment after disinfection. The samples were collected in sterile polypropylene bottles, shipped overnight on ice to the US EPA, Cincinnati, OH, and analyzed within 24 hours of collection.

E. coli titers were determined for all samples using membrane filtration method 9222 I (Standard Methods for the Examination of Water and Wastewater 2017). E. coli identification was confirmed using BBL Crystal Kits and Crystal Mind Software with the Autoreader (BD Bioscience, Sparks, MD). Confirmed E. coli isolates were then assigned a unique identification number and stored in 10% glycerol at −80 °C until further analysis.

The four antibiotics were selected because of the 2015 Centers for Disease Control and Prevention's definition for CRE, i.e., full resistance to two third generation cephalosporins and intermediate to full resistance to a carbapenem. Ciprofloxacin was chosen to ensure coverage for the H30R subclone within STc131. This subclone is known for its characteristic resistance to ciprofloxacin and for causing most multi-drug-resistant E. coli infections in the USA (Johnson et al. 2010).

Real-time PCR for carbapenemase and ESBL genes

CREC isolates were screened by real-time polymerase chain reaction (PCR) for four types of ESBL-encoding genes and five types of carbapanemase-encoding genes (Table 1). PCR was performed in a QuantStudio 6 Flex instrument (Applied Biosystems, Foster City, CA) as previously described (Ryu et al. 2012). Each PCR plate included controls to check for sample cross-contamination and positive controls. Dissociation curves were examined for evidence of potential primer-dimers and other non-specific reaction products.

Phylotyping and sequence typing

Phylogenetic groups of the AR E. coli isolates were defined by the quadraplex PCR method of Clermont et al. (2012). Clonal lineages were identified using 2-locus sequence analysis of fumC and fimH (CH typing) (Weissman et al. 2012). Each allele combination (i.e., CH type) was assigned to a putative sequence type (ST) based on the known association of CH types with STs (Weissman et al. 2012). In ambiguous situations, additional housekeeping gene loci were sequenced to clarify the most likely associated ST, according to Enterobase (http://enterobase.warwick.ac.uk).

RESULTS AND DISCUSSION

Titers of total E. coli, AR E. coli, and CREC were calculated for the WWTP samples to assess relative percentages of resistant organisms. Table 2 presents a summary of total E. coli titers along with the number of AR E. coli and CREC isolates obtained from each WWTP. Only results from the primary clarifiers are presented, as no AR E. coli isolates were obtained from the secondary effluents. Overall, 322 isolates were obtained that were resistant to ≥1 of the four antibiotics tested. Among the 322 AR E. coli isolates, the prevalence of resistance to individual agents declined sequentially as follows: cefotaxime (74%), ciprofloxacin (72%), ceftazidime (68%), and imipenem (20%). Additionally, 235 (73%) isolates qualified as multidrug-resistant, defined here as having resistance to ≥2 antibiotics tested. Notably, based on biochemical reactions, antibiotic resistance patterns, and sequence typing (results for the latter two are discussed below), and because no enrichment media were used, the AR isolates seemed unlikely to include replicates of the same clone from a given sample in the isolate collection.

Table 2

Summary of WWTP locations, Escherichia coli loads in primary clarifier effluents and antibiotic-resistant E. coli isolates obtained from each site

WWTP site

Average facility capacity

Dates of collection

Total E. coli (CFU/100 mL)

AR E. coli isolates

CR E. coli isolates

Raw wastewater composition

Ohio

13 MGD

July 2013

9.00 × 106

51

8

Domestic, urban

Sept. 2013

2.70 × 106

New Jersey

45 MGD

July 2013

3.50 × 106

84

22

Domestic, urban

Nov. 2012

4.50 × 106

N. California

33 MGD

July 2013

1.00 × 106

8

1

Agriculture, domestic, suburban

April 2013

TNTC

Texas

7.0 MGD

July 2013

1.50 × 106

14

1

Agriculture, domestic, rural

April 2013

2.00 × 107

Maryland

5.0 MGD

July 2013

3.00 × 107

32

8

Domestic, industrial, suburban

April 2013

5.50 × 107

Colorado

135 MGD

July 2013

7.50 × 106

29

10

Agriculture, domestic, industrial, urban

April 2013

3.00 × 107

S. California

275 MGD

July 2013

1.70 × 107

104

15

Industrial, agriculture, domestic, urban

Sept. 2013

1.00 × 107

WWTP site

Average facility capacity

Dates of collection

Total E. coli (CFU/100 mL)

AR E. coli isolates

CR E. coli isolates

Raw wastewater composition

Ohio

13 MGD

July 2013

9.00 × 106

51

8

Domestic, urban

Sept. 2013

2.70 × 106

New Jersey

45 MGD

July 2013

3.50 × 106

84

22

Domestic, urban

Nov. 2012

4.50 × 106

N. California

33 MGD

July 2013

1.00 × 106

8

1

Agriculture, domestic, suburban

April 2013

TNTC

Texas

7.0 MGD

July 2013

1.50 × 106

14

1

Agriculture, domestic, rural

April 2013

2.00 × 107

Maryland

5.0 MGD

July 2013

3.00 × 107

32

8

Domestic, industrial, suburban

April 2013

5.50 × 107

Colorado

135 MGD

July 2013

7.50 × 106

29

10

Agriculture, domestic, industrial, urban

April 2013

3.00 × 107

S. California

275 MGD

July 2013

1.70 × 107

104

15

Industrial, agriculture, domestic, urban

Sept. 2013

1.00 × 107

To estimate the overall prevalence of AR E. coli within the WWTP-associated E. coli population, colony counts for all 14 primary effluent samples were summed and an average was calculated for total E. coli and for E. coli resistant to imipenem, ciprofloxacin, cefotaxime, and ceftazidime. The average AR fraction within the total E. coli population, by agent, was 0.0022% for imipenem, 0.38% for cefotaxime, 0.031% for ceftazidime, and 3.1% for ciprofloxacin (Table S1, available with the online version of this paper). Comparable percentages were reported previously for raw wastewater from Poland, although no carbapenem-resistant organisms were found in that study (Łuczkiewwicz et al. 2010). It is difficult to compare the percentages of AR E. coli seen in wastewater to clinical studies, but percentages of CREC ranged from 0.7 to 1.9% in a survey of clinical infections, depending on the site of infection (Weiner et al. 2016). These higher carbapenem resistance percentages, as compared to the present findings in wastewater, would be expected because clinical settings will naturally concentrate AR isolates.

Antibiotic resistance in E. coli is more concerning when it occurs in strains capable of causing clinical infections, due to the potential for dissemination and the resulting morbidity and mortality. Phylogenetic analyses have shown consistently that most clinical E. coli isolates from human extraintestinal infections are derived from phylogroups B2 and D (Clermont et al. 2012). However, transfer of antibiotic resistance genetic elements from commensal to pathogenic strains is suspected to occur in reservoirs where these groups co-mingle, including WWTPs (Marshall et al. 2009; Bailey et al. 2010). Table 3 presents the phylotypes and associated resistance profiles of the present 322 AR E. coli isolates. Phylotypes B2 and D accounted collectively for nearly half (n = 181, 45%) of the sewage-source AR isolates (Table 3). Adding potentially pathogenic phylogroup F (Clermont et al. 2012) increased this to 56% of the total.

Percentage of total number of isolates resistant to specific antibiotic in parentheses.

Overall, 65/322 (20%) isolates were imipenem-resistant. CREC (n = 65) were concentrated in virulence-associated phylogroups B2, D and F (38/181 (21%)), as compared with the other phylogroups combined (27/141 (19%)). By contrast, all phylogroups had a high prevalence of multi-resistant isolates, including resistance specifically to ciprofloxacin and ESCs. On a percentage basis, phylogroup C had the highest percentages of total AR E. coli.

To further characterize the AR E. coli isolates, STs were determined to identify lineages associated with extraintestinal infections (Table 4). CH typing identified several ExPEC-associated clonal complexes (Johnson et al. 2010, 2017; Pitout 2012; Xia et al. 2017), namely (number of isolates, % of 322), STc131 (28, 8.7%), STc648 (17, 5.3%), ST1193 (25, 7.8%), and STc405 (12, 3.7%). Regarding the temporal and geographical distribution of these AR isolates from ExPEC-associated STs, carbapenem-resistant STc131 was identified in three separate samples from two locations (NJ, both summer and cooler temperatures; southern CA, summer only). Likewise, STc648 was identified in five locations, including two carbapenem-resistant isolates from two separate samples from one location (NJ, cooler and summer months); ST1193 was identified in five locations, including three CREC isolates from one sample in one location (NJ, summer); and STc405 was identified in five locations, including two CREC isolates from one sample in one location (MD, summer). These data indicate a widespread dispersal of these clinically significant AR STs with respect to locale and seasonality.

Table 4

Number of sequence types complexes found among AR E. coli known to be associated with clinically significant infections and their associated antibiotic resistance

Antibiotic resistance

B2-ST131-O25b and B2-ST131-O16

D-STc 405

STc 648

ST 1193

(n = 28)

(n = 12)

(n = 18)

(n = 27)

Imipenem

4 (14)

2 (17)

2 (11)

3 (11)

Ciprofloxacin

21 (75)

11 (92)

17 (94)

22 (81)

Ceftazidime

15 (54)

9 (75)

9 (50)

10 (37)

Cefotaxime

18 (64)

11 (92)

12 (67)

12 (44)

Antibiotic resistance

B2-ST131-O25b and B2-ST131-O16

D-STc 405

STc 648

ST 1193

(n = 28)

(n = 12)

(n = 18)

(n = 27)

Imipenem

4 (14)

2 (17)

2 (11)

3 (11)

Ciprofloxacin

21 (75)

11 (92)

17 (94)

22 (81)

Ceftazidime

15 (54)

9 (75)

9 (50)

10 (37)

Cefotaxime

18 (64)

11 (92)

12 (67)

12 (44)

Cell number represents number of isolates resistant to antibiotic, with percent in parentheses.

Determining the mechanisms that impart carbapenem resistance is imperative for epidemiological reasons. The major reason for carbapenem resistance is the presence of a carbapenemase-encoding gene. Many such genes are located on mobile genetic elements (Tait 1993), and tracking their dissemination can provide important information to health care professionals. Screening of our 65 CREC isolates by PCR for five carbapenemase genes and four ESBL genes (Table 5) showed that 45 (62%) of the isolates contained ≥1, and 20 (31%) contained ≥2, of the nine studied carbapenemase and ESBL genes, mainly VIM (36, 55%) and TEM (24, 37%), but also GES, SHV, IMP, OXA, KPC, CTX, and NDM. Few of these isolates harbored blaKPC (n = 2), and none carried blaNDM or blaOXA, which are the focus of multiple clinical case studies. The 20 (31%) remaining CREC isolates with no detected target gene conceivably could harbor antibiotic resistance genes not targeted in the study, or an alternate carbapenem resistance mechanism, such as loss of outer membrane proteins or efflux pumps/porin mutations, which have been demonstrated previously (Kong et al. 2018).

Samples were all negative for blaNDM, blaSHV, blaGES, and blaOXA genes.

The 36 blaVIM-containing CREC isolates were distributed by phylogroup as follows (% of 36): group D (19 isolates, 53%), B2 (6, 17%), C (5, 14%), B1 (3, 8%), and A (3, 8%). Thus, 70% of the blaVIM-positive isolates belonged to extraintestinal virulence-associated phylogroups B2 and D. As for distribution by ST, blaVIM occurred in three STc131 isolates (one of which also had blaKPC), one STc405 isolate, and one ST1193 isolate. The high prevalence of blaVIM in our study is notable considering the typical occurrence of this gene within integrons on broad-host-range plasmids. We are unaware of previously published data demonstrating such extensive prevalence of blaVIM among clinical E. coli isolates. Metallo-β-lactamases such as VIM were reported previously to be common worldwide, but rare in Enterobacteriaceae in the USA (Gupta et al. 2011; Nordmann et al. 2011).

CONCLUSIONS

This one-year study of water samples from seven geographically dispersed WWTPs in the USA identified 65 CREC isolates, of which 38 were from ExPEC-associated phlyogroups, including 11 from ExPEC-associated STs. Carbapenem-resistant STc131 was identified in three separate samples from two locations, and other clinically important STs were identified in multiple samples from other locations, demonstrating the plausible release of these organisms into receiving bodies of water.

Antibiotic resistance, including resistance to carbapenems, was not limited to pathogenic E. coli phylotypes or STs, as evidenced by the commensal phylotypes (A and C) also harboring multi-drug resistance patterns. Conceivably, this could be indicative of ectotherms harboring AR E. coli in their intestinal tracts as part of their normal microbiota.

The occurrence of CREC and other AR E. coli with potentially mobile resistance genes in wastewater is concerning because wastewater may serve as a reservoir for gene exchange. This implies a risk of AR E. coli and CREC being disseminated into the environment and thence back to humans, with routes of exposure potentially including contaminated recreational water and land application of biosolids. Our ability to understand the extent of this risk will depend, in part, on more detailed information concerning the fate of AR E. coli, including CREC, introduced in indigenous microbial communities, the rate of horizontal gene transfer in these communities, and how best to define the relative risk associated with both introduced and indigenous AR bacteria (Pepper et al. 2018).

Future studies should address the impact of multiple factors (e.g., geographic location, season, types of wastewater, and treatment plant configuration) on the prevalence of AR in wastewater. Additionally, rapid and reliable methods are needed to allow enumeration and characterization of AR organisms with respect to phylogenetic and clonal background, pathogenic potential, and resistance mechanisms. Identification of genetic factors that impart antibiotic resistance is essential when utilizing wastewater as an epidemiological screening tool for detection of emerging resistance patterns in comparison with clinical isolates from a given geographical locale.

ACKNOWLEDGEMENTS

The US Environmental Protection Agency (EPA), through its Office of Research and Development, funded and managed the research described herein. This work has been subjected to the agency's administrative review and has been approved for external publication. This work was also supported in part by Office of Research and Development, Department of Veterans Affairs (VA). Any opinions expressed in this paper are those of the authors and do not necessarily reflect the views of the EPA or VA; therefore, no official endorsement should be inferred. Any mention of trade names or commercial products does not constitute endorsement or recommendation for use. All the positive control strains for qPCR testing were graciously provided by Drs Johann Pitout and Gisele Pierano from the University at Calgary Medical School, Calgary, Alberta, Canada. We would also like to thank Connie Clabots and Steph Porter for their technical assistance.

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